Recently a variety of non-isotopic labeling methods have been developed to replace radioactive labels in DNA probe-based assays. It is most common in such methods to use marker enzymes to detect nucleic acid probes using either colormetric, chemiluminescent, bioluminescent or fluorescent methods. Each of these methods have been used reliably for both hybridization of DNA probe-based assays for nucleic acid detection as well as in solid-phase immunochemical a s says wherein the target molecule is typically an antigen of interest.
Regardless of the type of non-isotopic detection method used, the labels are typically measured directly with fluorophores (without use of enzymes) or indirectly using enzyme amplification schemes. A clear advantage of an indirect labeling scheme is the increased sensitivity one achieves through enzymatic amplification of the signal. However, a disadvantage of such methods as they are currently practiced in the field is that many steps are required in the assay protocol, requiring more time to complete the assay. Moreover, a greater number of reagents are required which means greater cost. In addition, where the method of detection is enzyme-based, the enzyme's activity, stability and its shelf life need to be considered if one is to expect optimum performance of the assay.
Chemiluminescence detection relies on a chemical reaction that generates light. It is this method which is now widely used for both nucleic acid detection as well as solid-based immunodetection due to its high sensitivity and wide variety of analysis methods ranging from manual film reading to instrumentation for processing images. Most commercially available chemiluminescent detection systems employ enzyme conjugates to increase detection sensitivity through amplification of the signal and, therefore, suffer from the same disadvantages described above.
In view of the simplicity of chemical reactions relative to enzymatic reactions, it would be desirable to achieve chemiluminescent signal amplification by chemical as opposed to enzymatic means. Moreover, non enzymatic systems have the advantage over enzyme-mediated systems of faster kinetics which result in peak light output within seconds. U.S. Pat. No. 5,516,636 to McCapra and a later publication by Schubert (Nucleic Acids Research, 1995, Vol. 23, No. 22 p. 4657) describe the use of sensitizer-labeled oligonucleotide probes for the detection of nucleic acid target molecules. In a solid phase DNA probe assay, a DNA target molecule is bound to a membrane and hybridized to a sensitizer-labeled oligonucleotide complementary in sequence to the target DNA. The membrane is subsequently contacted with an olefin. Upon exposure of the membrane to ambient oxygen and light, the sensitizer molecules become excited and transfer their excess energy to ambient oxygen for formation of singlet oxygen. The singlet oxygen therein produced reacts with the olefin on the membrane to form a stable 1,2-dioxetane in the area of the hybridization zone which when subsequently exposed to heat, chemical treatment or enzymatic treatment decomposes to emit light. Thus, oligonucleotides labeled with sensitizer are able to amplify the dioxetane concentration based on repeated excitation/oxygen quenching cycles to achieve a high level of sensitivity.
Prior art chemiluminescent assays employing sensitizers have generally required that a sensitizer-labeled probe hybridized to an analyte on a membrane be brought in contact with olefin for reaction to form a decomposable dioxetane. The membrane containing the analyte is then triggered by an activating source (e.g. base and/or heat) to produce a signal. The disadvantage of this format is that, because the analyte must be subjected to heat and/or base, it is not further utilized for additional testing and analysis.
U.S. Pat. No. 6,143,514 discloses a matrix having incorporated therein a label capable of being modified by a singlet oxygen and a non-photoactivatable catalyst (e.g. an enzyme) that is capable of catalyzing the formation of singlet oxygen from hydrogen peroxide. The catalyst coated matrix is incubated with assay medium suspected of containing hydrogen peroxide to permit the hydrogen peroxide to react with the catalyst to form singlet oxygen. The reaction of singlet oxygen with the label is determined, the reaction thereof indicating the presence of a compound capable of generating hydrogen peroxide. One disadvantage of this assay is that, because it is enzyme-based, the activity of the enzyme, its stability and its shelf-life need to be monitored as discussed above.
It would be advantageous to provide a method of performing a sensitizer-mediated solid phase chemiluminescent assay which would allow for reuse of a membrane-bound analyte. This is particularly desirable when amounts of available analyte for testing are limited. Such a method would preferably employ a membrane containing a solid olefin immobilized on or impregnated with, the membrane being suitable for use in both solid phase nucleic acid assays and immunoassays and, further, being able to be analyzed by methods ranging from manual film reading to instrumentation for processing images.